Characterization of Protein Kinase ULK3 Regulation by Phosphorylation and Inhibition by Small Molecule SU6668

Serine/threonine
protein kinase ULK3 is implicated in a variety of cellular processes,
including autophagy, cell division, and execution of the Sonic hedgehog
pathway. However, very little about how its biological activity could
be controlled is known. This study focuses on unraveling biochemical
insights into the mechanism of inhibition and activation of ULK3.
We identify novel phosphorylation sites in ULK3 and show that autophosphorylation
has no impact on the kinase activity of the protein. We further demonstrate
that phosphorylation of two residues in the kinase domain of ULK3
by an as yet unidentified kinase may completely abolishes its catalytic
activity. We show that a low-molecular weight inhibitor SU6668, designed
as an ATP competitive inhibitor for tyrosine kinases, binds in the
ATP pocket of ULK3 yet inhibits ULK3 kinase activity in a partially
ATP noncompetitive manner. Finally, we demonstrate that the ULK3 kinase
domain, annotated in silico, is not sufficient for
its kinase activity, and additional amino acids in the 271–300
region are required.